Our attack on the aromatic amino acid hydroxylases and the enzymes required for synthesis and maintenance of the cofactor, tetrahydrobiopterin, has been multi-level. We have cloned several of the cDNAs for enzymes in this system. These clones comprise a battery of probes to scan DNA or RNA samples from individuals with mental diseases to identify unknown mutations in genes that may be responsible for some subset of these disorders. Additionally, our cloning and expression of these enzymes has produced quantities of protein suitable for biochemical study. We are applying all that is known about catalytic and regulatory sites to these enzymes and have expressed several enzymes carrying directed alterations. In this manner, we have been testing our ideas about significant domains to determine the roles that these sites play in catalysis, affinity to substrates and effector molecules, etc. For example, we have expressed our cDNA clone for rat sepiapterin reductase, an enzyme essential for synthesis of the cofactor tetrahydrobiopterin (essential for the hydroxylation of aromatic amino acids), in bacteria and obtained a large quantity of active enzyme. This represents the first clone (from any organism) for any enzyme in the tetrahydrobiopterin biosynthetic pathway, which is necessary for the biosynthesis of dopaminergic and serotinergic neurotransmitters.